The Human Immunodeficiency Virus (HIV) is a human retrovirus which is the etiologic agent of the Acquired Immunodeficiency Syndrome. To study the cellular factors involved in the transcriptional regulation of this virus, we performed DNAase I footprinting of the viral LTR. Five regions of the viral LTR appear critical for DNA binding of cellular proteins. There are four upstream regulatory elements and a down-stream element which includes a portion of the untranslated region of the LTR. The binding characteristics of these factors do not appear to be altered in HIV infected extracts compared to mock extracts. Mutations of these binding domains have significant effects on the basal level of transcription and the ability to be induced by the viral tat protein. One upstream protected region has significant sequence homology with elements found in the SV40, the immunoglobulin kappa gene, and the cytomegalovirus enhancers. The factor binding to this enhancer region has increased binding activity and altered binding characteristics in lymphoid cells. The cooperative interactions between several distinct cellular proteins are required for the transcriptional regulation of the viral LTR. The goal of this grant is to better understand the role these proteins have in the transcriptional regulation of HIV. Oligonucleotide-directed mutagenesis will be used to characterize the binding domains in the LTR. Each binding protein will be purified by a combination of column chromatography and oligonucleotide affinity chromatography. We will then determine whether cell specific modifications of these proteins occur and whether HIV infection alters the characteristics of these proteins. Finally, the role of each protein in in vitro transcriptional assays will be determined. A detailed characterization of these cellular transcription factors should provide important insights into the mechanisms of HIV gene regulation.